Expression Characteristics Of Transcription Factor FOSB In Lung Carcinogenesis Induced By Polycyclic Aromatic Hydrocarbons And Its Influencing Mechanisms On The Malignant Progression And Prognosis In Lung Cancer In A P53-Dependent Manner

Objective: Lung cancer is the leading cause of cancer morbidity and mortality worldwide and is imposing a huge burden on public health.Effective early diagnosis is determined as an important prevention and control goal to reduce lung cancer-related deaths and improve the prognosis of patients.Platinum-based chemotherapy remains the first-line treatment for lung cancer,but the occurrence of adverse clinical events such as frequent drug resistance,progression,and recurrence poses a great challenge to the prognosis of lung cancer.Therefore,an in-depth study on the mechanisms underlying lung cancer development to identify the key molecular events involved and an active exploration on potential biomarkers or intervention targets to help with diagnosis or prognosis,are both of great public health significance to achieving the tertiary prevention of lung cancer.Tobacco smoking remains the major risk factor for lung cancer,and polycyclic aromatic hydrocarbons(PAHs)carcinogens represented by the benzo(a)pyrene in tobacco smoke are most closely related to the development of lung cancer.Lung cancer is generally considered to be resulted from both environmental and genetic factors.Genome-wide association studies and epidemiological evidence have demonstrated that genetic or epigenetic variations in band 3 of region 1 of the long arm of human chromosome 19(19q13)are strongly associated with the onset risk and prognostic outcome of lung cancer.The FBJ Murine Osteosarcoma Viral Oncogene Homolog B(FOSB)is one of the genes located at 19q13,which encodes a protein that functions biologically as a nuclear transcription factor.However,the roles of FOSB in the development and prognosis of lung cancer have been being under controversy in recent years,and the specific mechanisms have not yet been fully elucidated.In our preliminary study,we found that FOSB expression was significantly downregulated in PAHs-induced malignant transformation of bronchial epithelial cells and smoking-associated lung cancer tissues,suggesting its possible involvement as a potential tumor suppressor in tobacco smoke exposure-induced lung carcinogenesis.Interestingly,the further survival analysis showed that FOSB expression in lung cancer tissues with different mutation status of the TP53 gene indicated exactly opposite prognoses,reminding that TP53 mutation status might act as a key molecular switch leading to a shift in the tumor biological effects of FOSB in lung cancer cells.Thereupon,the present study proposed that it was necessary to systematically explore the expression characteristics of the transcription factor FOSB in PAHs-induced lung carcinogenesis and its specific roles and potential molecular mechanisms in the malignant progression and the prognosis of platinum-based chemotherapy in lung cancer under a specific genetic context of the mutation status of the TP53 gene,in order to deliver new scientific clues and unique mechanistic insights for the identification of more precise biomarkers and potential therapeutic targets for the susceptibility,diagnosis and prognosis in smoking-associated lung cancer.Methods: 1.Based on a BPDE-induced malignant transformation model of human bronchial epithelial cells 16 HBE previously established by our research group,RTq PCR and Western Blot were used to detect the changes in FOSB m RNA and protein expression levels during the malignant transformation.2.RT-q PCR and Western Blot were used to detect the differential expression in FOSB m RNA and protein between human normal lung epithelial cell lines and non-small cell lung cancer(NSCLC)cell lines.3.The NSCLC cohort acquired from the TCGA database was used to analyze the differential expression in FOSB m RNA between smoking-associated NSCLC tissues and paired paraneoplastic tissues,and the correlations of FOSB expression with the clinicopathological characteristics and prognosis in NSCLC patients.4.RT-q PCR and Western Blot were used to detect the differential expression in FOSB m RNA and protein between clinically collected smoking-associated lung cancer tissues and paired paraneoplastic tissues.5.FOSB,p53-WT,p53-R175 H,p53-R248 W,p53-R248 L,p53-R248 Q,p53-R273 H and p53-R273 L overexpression plasmids were constructed using molecular cloning technique(OBi O Technology Co.,Ltd.,China),and the agarose gel electrophoresis followed by Sanger sequencing(Sangon Biotech Co.,Ltd.,China)was conducted to verify the mutant sites of the products.6.H1299 lung cancer cells with p53 deficiency were transfected with the wild-type p53 or different site-specific mutant p53 plasmids respectively,and the m RNA and protein expression levels of p53,together with its downstream targets P21 and PUMA,were detected by RT-q PCR and Western Blot.7.H1299 lung cancer cells expressing wild-type p53 or different site-specific mutant p53 were transfected with the FOSB overexpression plasmid,and its m RNA and protein expression levels were detected by RT-q PCR and Western Blot.8.CCK-8assay was used to detect the effects of FOSB overexpression on the proliferation ability in H1299 cells carrying different mutation types of TP53.9.Colony formation assay was used to detect the effects of FOSB overexpression on the independent viability in H1299 cells carrying different mutation types of TP53.10.Wound healing assay and Transwell migration assay were used to detect the effects of FOSB overexpression on the migration ability in H1299 cells carrying different mutation types of TP53.11.Matrigel-coated Transwell invasion assay was used to detect the effects of FOSB overexpression on the invasion ability in H1299 cells carrying different mutation types of TP53.12.CCK-8 assay was used to detect the effects of FOSB overexpression on the cisplatin sensitivity in H1299 cells carrying different mutation types of TP53.13.Flow cytometry was used to detect the effects of FOSB overexpression on cisplatininduced apoptosis in H1299 cells carrying different mutation types of TP53.14.H1299(p53-Null),H1299(p53-WT)and H1299(p53-R248Q)cells with FOSB overexpression were constructed followed by the transcriptome sequencing(Lianchuan Biotech Co.,Ltd.,China),and the differentially expressed genes(DEGs)were visualized by volcano maps.15.Biological enrichment analysis(GO analysis)was conducted using the set of DEGs upregulated in the above three transfected cells with FOSB overexpression,and the results of the enrichment analysis were visualized by bubble plots.16.RT-q PCR and Western Blot were used to detect the effects of FOSB overexpression on the candidate transcriptional targets of FOSB and their downstream signaling pathways in H1299(p53-Null),H1299(p53-WT),A549(p53-WT),H1299(p53-R248Q)and PC-9(p53-R248Q)cells.17.Changes in tumor biological phenotypes were detected after the candidate transcriptional targets of FOSB were silenced by si RNA-mediated RNA interference technology: Cell proliferation ability,evaluated by the CCK-8 assay;Cell independent viability,evaluated by the colony formation assay;Cell migration ability,evaluated by the wound healing assay and Transwell migration assay;Cell invasion ability,evaluated by the Matrigel-coated Transwell invasion assay;Cell sensitivity to cisplatin,evaluated by the CCK-8 assay.18.Endogenous wild-type p53 in A549 cells was silenced by si RNA-mediated RNA interference,and RT-q PCR and Western Blot were used to detect the effects of FOSB overexpression on the m RNA and protein levels of downstream indicators involved.19.Endogenous mutant p53-R248 Q in PC-9 cells was silenced by si RNA-mediated RNA interference,and RT-q PCR and Western Blot were used to detect the effects of FOSB overexpression on the m RNA and protein levels of downstream indicators involved.20.The Gene MANIA database was used to establish a protein-protein interaction network with FOSB and p53 as the central hub.21.The Co-IP assay was conducted to detect the potential protein-protein interactions between FOSB and the endogenous or exogenous wild-type p53 or mutant p53-R248 Q in H1299(p53-WT),A549(p53-WT),H1299(p53-R248Q)and PC-9(p53-R248Q)cells.22.The JASPAR database was used to predict potential binding sites for the transcription factor FOSB in the promoters of the PREX1,IGFBP5,AKR1C3 and ALDH3A1 genes.23.The Ch IP-q PCR assay was used to confirm the direct binding of the transcription factor FOSB to the promoter sequences in the candidate target genes PREX1,IGFBP5,AKR1C3 and ALDH3A1,and the effects of changes in p53 status on the binding capacity of FOSB to the promoters of the above candidate target genes.Results: 1.Molecular characteristics of transcription factor FOSB expression in BPDEinduced malignant transformation of lung epithelial cells and smoking-associated lung cancer tissues:(1)FOSB m RNA and protein expression levels were significantly downregulated in BPDE-induced malignant transformation of lung epithelial cells,NSCLC cell lines,and smoking-associated NSCLC tissues(P<0.05).(2)FOSB expression indicated different prognoses in lung adenocarcinoma(LUAD)and lung squamous cell carcinoma(LUSC): FOSB high expression in smoking-associated LUAD indicated longer overall survival(OS)and was associated with positive clinicopathological characteristics(P<0.05),whereas the opposite was true in smokingassociated LUSC(P<0.05).(3)There was a significant difference in the mutation frequency of the TP53 gene in NSCLC: it was about 50% in LUAD while more than85% in LUSC.(4)The prognostic effects of FOSB expression in NSCLC were dependent on the mutation status of the TP53 gene: In the genetic background of wildtype TP53,FOSB high expression indicated longer OS and was associated with positive clinicopathological characteristics and prognostic indices(P<0.05);while in the genetic background of mutant TP53,FOSB high expression indicated shorter OS and was associated with poor clinicopathological characteristics and prognostic indices(P<0.05).2.Effects of transcription factor FOSB on the malignant biological behaviors and cisplatin sensitivity in H1299 lung cancer cells carrying different mutation types of TP53:(1)In H1299 cells with TP53 deficiency(TP53-Null),FOSB overexpression promoted cell proliferation(P<0.05)and colony-forming ability(P<0.05);in H1299 cells carrying wild-type TP53(TP53-WT),FOSB overexpression inhibited cell proliferation(P<0.05)and colony-forming ability(P<0.05);in H1299 cells carrying mutant TP53-R175 H,TP53-R248 Q and TP53-R273 L,FOSB overexpression promoted cell proliferation(P<0.05)and colony-forming ability(P<0.05).(2)In H1299 cells with TP53 deficiency(TP53-Null),FOSB overexpression promoted cell migration and invasion abilities(P<0.05);in H1299 cells carrying wild-type TP53(TP53-WT),FOSB overexpression inhibited cell migration and invasion abilities(P<0.05);in H1299 cells carrying mutant TP53-R175 H,TP53-R248 Q and TP53-R273 L,FOSB overexpression promoted cell migration and invasion abilities(P<0.05).(3)In H1299 cells carrying wild-type TP53(TP53-WT),FOSB overexpression enhanced the cisplatin sensitivity in tumor cells(P<0.05);while in H1299 cells carrying mutant TP53-R248 Q,FOSB overexpression reduced the cisplatin sensitivity in tumor cells(P<0.05).(4)In H1299 cells carrying wild-type TP53(TP53-WT),FOSB overexpression promoted cisplatininduced apoptosis in tumor cells(P<0.05);while in H1299 cells carrying mutant TP53-R248 Q,FOSB overexpression inhibited cisplatin-induced apoptosis in tumor cells(P<0.05).3.Molecular mechanisms underlying the regulation of malignant biological behaviors and cisplatin sensitivity in lung cancer cells by FOSB in a p53-dependent manner:(1)FOSB overexpression led to unique transcriptomic changes in three lung cancer cells with different mutation status of TP53,namely H1299(p53-Null),H1299(p53-WT)and H1299(p53-R248Q).(2)Biological enrichment analysis combined with the RT-q PCR assay identified PREX1 as a specific transcriptional target of FOSB in lung cancer cells with TP53 deficiency(P<0.05),IGFBP5 as a specific transcriptional target of FOSB in lung cancer cells carrying TP53-WT(P<0.05),and AKR1C3 and ALDH3A1 as specific transcriptional targets of FOSB in lung cancer cells carrying TP53-R248Q(P<0.05).(3)In lung cancer cells with TP53 deficiency,FOSB overexpression specifically upregulated PREX1(P<0.05)and activated its downstream RAC1-ERK/AKT oncogenic signaling pathway(P<0.05),while PREX1 knockdown(P<0.05)interdicted the activation of the RAC1-ERK/AKT oncogenic signaling pathway by FOSB overexpression(P<0.05);in lung cancer cells carrying TP53-WT,FOSB overexpression specifically upregulated IGFBP5(P<0.05)and activated its downstream p53 signaling pathway(P<0.05)and inhibited the ERK/AKT oncogenic signaling pathway(P<0.05),while IGFBP5 knockdown(P<0.05)interdicted the activation of the p53 signaling pathway(P<0.05)and the inhibition of the ERK/AKT oncogenic signaling pathway(P<0.05)by FOSB overexpression;in lung cancer cells carrying TP53-R248 Q,FOSB overexpression specifically upregulated AKR1C3(P<0.05)and ALDH3A1(P<0.05),and activated their downstream ERK/AKT oncogenic signaling pathway(P<0.05),while AKR1C3 knockdown(P<0.05)interdicted the activation of the ERK/AKT oncogenic signaling pathway by FOSB overexpression(P<0.05).(4)FOSB overexpression significantly promoted the proliferation,migration and invasiveness in lung cancer cells with TP53 deficiency(P<0.05),while PREX1 knockdown interdicted the promotion of the malignant biological behaviors in lung cancer cells by FOSB overexpression(P<0.05);FOSB overexpression significantly inhibited the proliferation,migration and invasiveness and enhanced the cisplatin sensitivity in lung cancer cells carrying TP53-WT(P<0.05),while IGFBP5 knockdown interdicted the inhibition of the malignant biological behaviors(P<0.05)and the promotion of the cisplatin sensitivity(P<0.05)in lung cancer cells by FOSB overexpression;FOSB overexpression significantly promoted the proliferation,migration and invasiveness and reduced the cisplatin sensitivity in lung cancer cells carrying TP53-R248Q(P<0.05),while AKR1C3 knockdown interdicted the promotion of the malignant biological behaviors(P<0.05)and the reduction of the cisplatin sensitivity(P<0.05)in lung cancer cells by FOSB overexpression.(5)Silencing of endogenous p53-WT in A549 cells resulted in a shift in the transcriptional targets of FOSB from IGFBP5 to PREX1(P<0.05);silencing of endogenous p53-R248 Q in PC-9 cells resulted in a shift in the transcriptional targets of FOSB from AKR1C3 and ALDH3A1 to PREX1(P<0.05).(6)The Co-IP assay demonstrated direct protein-protein interactions between FOSB and endogenous or exogenous p53-WT or p53-R248 Q by immunoprecipitation using either FOSB-specific antibody or p53-specific antibody.(7)In H1299(p53-Null)lung cancer cells,only the direct binding of FOSB to the PREX1 promoter was detected(P<0.05);in H1299(p53-WT)lung cancer cells,p53-WT overexpression weakened the binding of FOSB to the PREX1 promoter(P<0.05)and facilitated its binding to the IGFBP5 promoter(P<0.05);in H1299(p53-R248Q)lung cancer cells,p53-R248 Q overexpression weakened the binding of FOSB to the PREX1 promoter(P<0.05)and facilitated its binding to the AKR1C3(P<0.05)and ALDH3A1(P<0.05)promoters.Conclusion: The transcription factor FOSB is significantly down-regulated in polycyclic aromatic hydrocarbon-induced malignant transformation of lung epithelial cells and smoking-associated lung cancer tissues,but its expression indicates a positive prognosis in patients carrying wild-type TP53 while a poor one in those carrying mutant TP53.In the genetic background of TP53 deficiency,transcription factor FOSB promotes the malignant biological behaviors in lung cancer cells by regulating the downstream RAC1-ERK/AKT oncogenic signaling pathway through PREX1;in the genetic background of wild-type TP53,it suppresses the malignant biological behaviors in lung cancer cells and enhances their cisplatin sensitivity by regulating the downstream p53 signaling pathway and the ERK/AKT oncogenic signaling pathway through IGFBP5;in the genetic background of mutant TP53-R248 Q,it promotes the malignant biological behaviors in lung cancer cells and reduces their cisplatin sensitivity by regulating the downstream ERK/AKT oncogenic signaling pathway through AKR1C3/ALDH3A1.By establishing direct protein-protein interactions with FOSB,wild-type or mutant p53 guides FOSB to recognize and bind to different promoter sequences to transcriptionally activate the expression of specific target genes,thereby having diverse influences on the progression and prognosis in lung cancer.